Culty M, Davidson M M, Haslam R J
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Eur J Biochem. 1988 Feb 1;171(3):523-33. doi: 10.1111/j.1432-1033.1988.tb13821.x.
Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)
在含有氯化锰的低葡萄糖台氏液中，将人血小板与肌醇 - [³H]肌醇一起孵育，可使磷酸肌醇的标记增加约7倍，并极大地促进了通过磷脂酶C激活形成的[³H]肌醇磷酸的测量。通过高压放电使标记的血小板通透化，并在0℃下用ATP、Ca²⁺缓冲液和鸟嘌呤核苷酸平衡，然后在有无凝血酶的情况下进行孵育。在有或无Ca²⁺离子存在下，将这些血小板与ATP一起孵育，会导致[³H]磷脂酰肌醇转化为[³H]磷脂酰肌醇4 - 磷酸和[³H]磷脂酰肌醇4,5 - 二磷酸（[³H]PtdInsP2）。在pCa为6时，添加100μM GTP[γS]既阻止了[³H]PtdInsP2的这种积累，又刺激了其分解；[³H]肌醇磷酸的形成增加了9倍。5分钟后，这些产物包括70%的[³H]肌醇单磷酸（[³H]InsP）、28%的[³H]肌醇双磷酸（[³H]InsP2）和2%的[³H]肌醇三磷酸（[³H]InsP3）。在较短的孵育时间内，发现[³H]InsP2和[³H]InsP3的百分比更高。在不添加Ca²⁺的情况下，[³H]肌醇磷酸的形成减少了90%以上。在10 mM Li⁺存在下，将通透化的血小板与GTP[γS]一起孵育，会降低[³H]InsP的积累并增加[³H]InsP2的积累，而不影响[³H]InsP3的水平。添加未标记的InsP3会降低外源[³²P]InsP3的细胞内水解，但不会捕获额外的[³H]InsP3。这些结果以及[³H]肌醇磷酸形成的时间进程表明，GTP[γS]刺激了磷脂酶C对[³H]磷脂酰肌醇4 - 磷酸池的作用，否则该池会转化为[³H]PtdInsP2，并且[³H]磷脂酰肌醇水解为[³H]InsP或[³H]PtdInsP2水解为[³H]InsP3的情况要少得多。在pCa为6时，向通透化的血小板中添加凝血酶（2单位/ml）会使[³H]InsP和[³H]InsP2的形成略有增加。凝血酶的这种作用在10 - 100μM GTP存在下增强了两倍，在4 - 40μM GTP[γS]存在下增强得更显著。在后者存在的情况下，凝血酶也会增加[³H]InsP3。用凝血酶和GTP[γS]孵育的通透化血小板形成的[³H]肌醇磷酸的总量与单独向完整血小板中添加凝血酶时观察到的量相当。然而，对形成的[³H]肌醇磷酸进行HPLC分析表明，通透化血小板中积累的[³H]InsP中约有75%是4 - 磷酸，而在凝血酶刺激的完整血小板中，高达80%是1 - 磷酸。（摘要截短至400字）
